Abstract With our unique multi-component clinical sample and advances in Mass Spectrometry (MS), we hope to identify a combination of multiple biomarkers that provide discriminatory and accurate biomarkers of Alzheimer?s disease (AD) to advance earlier detection and treatment. Currently, the ratio of tau/A? in cerebrospinal fluid (CSF) is the best available biomarker for separating AD from normal subjects and predicting disease progression, but its sensitivity and specificity are modest. Our co-investigator (co-I), Dr. Lu, discovered that a pathologic conformation, the cis-form of phosphorylated pT231-tau (cis-ptau), contributes to AD pathogenesis. Similarly, oligomeric A? (oA?) is the most toxic form of A? to neurons, and we found that oA? was increased in plasma and brain tissue of AD patients. Our hypothesis is that detection of increased levels of these pathologic species (cis-ptau and oA?) will yield a robust biomarker for AD. Our team is uniquely positioned to test this hypothesis. We are the first group who reported MS-based proteomic profiling of three dimensional (3D) neuronal culture. We have analyzed candidate biomarkers from the plasma, induced pluripotent stem cells (iPSC) derived neurons, and post-mortem brain tissue from the same AD patients, which is unprecedented. We have analyzed brain tissue from 10 cases out of 20 AD, 20 MCI and 20 non-demented (ND) subjects from VA Bedford/Boston University Alzheimer Disease Center (ADC) Brain Bank (directed by co- I, Dr. Stein). Our biomarker detection capability is well-developed and we have in hand a clinically well- characterized set of plasma, CSF, and brain samples. We will validate novel biomarkers using the test samples of plasma and CSF from the same patients with early and moderate AD (20 patients) and 20 control ND subjects from Wisconsin ADC (samples are currently stored in co-I Dr. Lu?s lab). We will first determine whether CSF cis-ptau alone sufficiently separates AD from ND subjects. Next, truncated A? peptides will be measured along with cis-ptau to determine whether ratios of cis-ptau/A? in CSF provides a better separation of AD from ND subjects. Finally, we will quantify plasma cis-ptau and plasma oA?, and use a combination of plasma cis-ptau and oA? along with CSF cis-ptau as biomarkers for AD.